Our objective is to develop a laminin isoform as an intravenously delivered protein therapy for treatment of Congenital Muscular Dystrophy Type IA (MCD1A). MCD1A is the most common form of congenital muscular dystrophy and is characterized by hypotonia, elevated serum CK, delayed motor milestones, respiratory insufficiency, feeding difficulties, joint contractures and scoliosis, and in some patients mental retardation and epilepsy. MCD1A is caused by a deficiency of the alpha2 chain present in laminin-211 and -221 (LAM-211/221), a large multifunctional extracellular matrix protein normally present in the basal lamina of skeletal muscle, cardiac and neuronal tissues. One major etiological basis of MCD1A is a loss of cellular adhesion;the absence of LAM-211 in skeletal and cardiac muscle results in loss of the crucial stabilizing link between the intracellular contractile apparatus, the muscle membrane, and extracellular basal lamina, and as a consequence muscles become abnormally sensitive to damage. A substantial delay in muscle regeneration occurs when skeletal muscle degeneration of dyW is provoked by notexin, indicating that an additional pathological component of laminin alpha2 chain deficiency is a defect in muscle regeneration. To prevent muscle instability and the ensuing pathology, a therapeutic intervention for MCD1A must restore the missing linkage between the contractile apparatus, muscle membrane and basal lamina. The most effective near-term therapies for MCD1A patients are likely to include the intravenous delivery of functionally redundant components of the basal lamina such as mini-agrins and the laminin isoform, small molecules that increase expression of the same, and molecules that alleviate the secondary manifestations of MCD1A. The laboratory of our academic partner Dr. Dean J. Burkin has generated preliminary data demonstrating that a single intramuscular or systemic dose of a laminin isoform to mdx mice, the mouse model of Duchenne muscular dystrophy, distributed to all skeletal and cardiac muscles, remained localized around myofibers for at least 4 weeks, substantially reduced myofiber degeneration, was associated with increased expression of the alpha7 integrin, and reduced serum creatine kinase activity. Subsequent preliminary data has demonstrated that multiple systemic doses of 1.0 mg/kg of the laminin isoform to dyW mice, the mouse model of MCD1A, result in distribution of the laminin isoform around skeletal myofibers, improved muscle morphology, reduced centrally nucleated myofibers, and improved health. Given that the laminin isoform can be delivered systemically to skeletal muscles of mdx and dyW mice, intravenous delivery of the laminin isoform would represent a powerful therapeutic for treatment of human MCD1A. Although the benefit of mini-agrins on dyW muscle regeneration has been established, the benefit of exogenous application of the laminin isoform on dyW muscle regeneration has yet to be established. The first specific aim of this proposal will ask if intramuscular delivery of the laminin isoform to dyW mice ameliorates the regeneration defect observed following cardiotoxin-provoked muscle degeneration. The second specific aim will expand upon our preliminary data and ask if chronic systemic administration of the laminin isoform improves the overall health of dyW mice. This will include assessment of more disease endpoints including weight gain, lifespan, locomotor activity and grip strength, creatine kinase, apoptosis, and histopathological changes. We will also test whether such effects correlate with an enhanced sarcolemmal expression of alpha7 integrin and whether anti-laminin isoform antibodies appear and whether they neutralize the laminin isoform. A successful demonstration that the laminin isoform is a safe and effective treatment for dyW/MDC1A will justify the construction of stable cell lines for the production of the recombinant human laminin isoform, as well as the attendant quality control assays to verify the structure, purity, bioactivity, and stability of the recombinant human laminin isoform. We have prepared and distributed a request for proposal (RFP) to various contract manufacturing organizations (CMO) for the production of large quantities of GLP-grade of the recombinant human laminin isoform. Upon further funding the recombinant human laminin isoform will be used for IND-enabling pharmacology and toxicology studies, and given approval a phase I and II clinical trial. The end result of our efforts will be an intravenously delivered protein therapy for patients with MCD1A, DMD and LGMD2C-F. PUBLIC HEALTH RELEVANCE: Congenital muscular dystrophy type 1A (MCD1A) is a progressively debilitating genetic disease and is the most common congenital muscular dystrophy. We are investigating a protein therapy that may stabilize skeletal muscles and improve muscle regeneration of MCD1A patients and improve their quality of life.